Plasma separation device

ABSTRACT

Devices and methods are provided that permit efficient and selective separation of liquid biological specimens into at least two constituent components to facilitate subsequent quantitative and qualitative analysis on at least one analyte of interest in at least one of the components. The devices generally include one or more sample deposition regions supported on a base. Each sample deposition region includes a separation membrane for separating the liquid biological specimen into two different fractions. The first fraction is trapped by the separation membrane while the second fraction passes through the separation membrane and into a respective collection membrane. The separation and collection membranes are easily separable from the devices and can be utilized for further processing and analysis.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/515,439, filed Jul. 18, 2019, now U.S. Pat. No. 10,690,654, which isa continuation of PCT Application No. PCT/US2018/015969, filed Jan. 30,2018, which claims the benefit of U.S. Provisional Patent ApplicationNo. 62/451,945, filed on Jan. 30, 2017. The entire contents of which areincorporated by reference herewith.

FIELD OF INVENTION

The present disclosure generally relates to devices and methods thatpermit efficient and selective separation of liquid biological specimens(e.g. biological fluids or biological specimen containing suspensions)into at least two constituent components to facilitate subsequentquantitative and qualitative analysis on at least one analyte ofinterest in at least one of the components.

BACKGROUND OF THE INVENTION

Biological specimens are often collected for analysis of the levels andconcentrations of various analytes contained therein. Although manydiagnostics are carried out on biological specimens in their nativestate, many times the biological specimen must be separated into itsconstituent components for a variety of reasons. Separating a biologicalspecimen into different constituent parts can maximize the precision,accuracy, and reproducibility of detecting and quantifying analytes ofinterest within the biological specimen. For example, it is oftennecessary to filter out solid components from whole blood (e.g., whiteblood cells, red blood cells, etc.), separate blood serum from wholeblood, and separate blood plasma from whole blood, to improve not onlythe recovery of select analytes from the biological specimen (e.g.viruses, plasma proteins, cytokines, chemokines, immunglobins, etc.) butalso improve the subsequent detection and analysis of those analytes. Asone example, red blood cells (erythrocytes) scatter and absorb lightand, therefore, can adversely affect diagnostic tests that rely onmeasurements of either reflected or transmitted light. Removing redblood cells can help obtain the most accurate reading possible.

Traditionally, liquid biological specimens have been separated bycentrifugation. For example, blood plasma and serum have been separatedfrom whole blood by centrifuging either before (for plasma) or after(for serum) clotting. However, centrifugation requires electricity andexpensive equipment that may not be readily available in a clinicallaboratory or out in the field. Further, centrifugation can damageanalytes of interest (e.g. nucleic acids such as DNA and RNA).

A number of techniques have been devised to avoid this problem. Thetechniques generally utilize a filtering device that separates a liquidbiological specimen into various components. However, these devices haveproven to be unsuitable for a variety of reasons. Therefore, what areneeded are improved devices and methods that permit efficient andselective separation of liquid biological specimens into at least twoconstituent components to facilitate subsequent quantitative andqualitative analysis on at least one analyte of interest in at least oneof the components.

SUMMARY OF THE INVENTION

This disclosure generally provides liquid biological specimen separationdevices and methods of using the same. In some aspects, a liquidbiological specimen separation device is provided that comprises a base;a collection membrane disposed on the base, a separation membranedisposed on the collection membrane; and a cover disposed on theseparation membrane, wherein the cover comprises an aperture thereinconfigured to allow deposition of a liquid biological specimen. In otheraspects, a liquid biological specimen separation device is provided thatcomprises a base; a plurality of collection membranes disposed on thebase; a plurality of separation membranes, wherein each separationmembrane is disposed on a corresponding one of the collection membranes;and a plurality of covers, wherein each cover is disposed on acorresponding one of the separation membranes, and wherein each covercomprises an aperture therein configured to allow deposition of a liquidbiological specimen therethrough. In other aspects, a liquid biologicalspecimen separation device is provided that comprises a base; acollection membrane disposed in the base, a separation membrane disposedin the base and on the collection membrane, and a cap disposed on theseparation membrane and base, wherein the cap comprises an aperturetherein configured to allow deposition of a liquid biological specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the drawings, which are meant to be exemplary and notlimiting, and wherein like elements are numbered alike. The detaileddescription is set forth with reference to the accompanying drawingsillustrating examples of the disclosure, in which use of the samereference numerals indicates similar or identical items. Certainembodiments of the present disclosure may include elements, components,and/or configurations other than those illustrated in the drawings, andsome of the elements, components, and/or configurations illustrated inthe drawings may not be present in certain embodiments.

FIGS. 1A-1B show a liquid specimen separation device in accordance withone example embodiment of the disclosure.

FIG. 2 shows an exploded view of a liquid specimen separation device inaccordance with one example embodiment of the disclosure.

FIGS. 3A-3B show a liquid specimen separation device in accordance witha second example embodiment of the disclosure.

FIGS. 4A-4B show a liquid specimen separation device in accordance witha third example embodiment of the disclosure.

FIG. 5 shows an exploded view of a liquid specimen separation device inaccordance with a third example embodiment of the disclosure.

FIG. 6 shows an exploded view of a liquid specimen separation device inaccordance with a fourth example embodiment of the disclosure.

FIG. 7 shows cut away view of a liquid specimen separation device inaccordance with a fourth example embodiment of the disclosure.

FIG. 8 shows a liquid specimen separation device in accordance with afourth example embodiment of the disclosure.

FIGS. 9A-9D show a liquid specimen separation device in accordance witha fifth example embodiment of the disclosure.

FIGS. 10A-10B show a liquid specimen separation device in accordancewith a sixth example embodiment of the disclosure.

FIGS. 11A-11F show a liquid specimen separation device in accordancewith a seventh example embodiment of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Devices and methods are provided that permit efficient and selectiveseparation of liquid biological specimens into at least two constituentcomponents to facilitate subsequent quantitative and qualitativeanalysis on at least one analyte of interest in at least one of thecomponents. The devices and methods fulfill the need for a convenientand simple method for filtering, separating, and/or storing an analyteof interest.

Examples of biological specimen suitable for use with devices describedherein include whole blood, plasma, urine, saliva, sputum, semen,vaginal lavage, bone marrow, breast milk, and cerebrospinal fluid. Oneadvantage of the present devices is that they can sufficiently preserveanalytes of interest.

The devices generally include one or more sample deposition regionssupported on an ergonomically configured base that permits ease ofhandling. Each sample deposition region includes a separation membranefor separating the liquid biological specimen into two differentfractions. The first fraction is trapped by the separation membranewhile the second fraction passes through the separation membrane andinto a respective collection membrane. The separation and collectionmembranes are easily separable from the devices and can be utilized forfurther processing and analysis. The devices can include other featuressuch as one or more covers (e.g. stickers) or caps that define thesample deposition regions, secure the separation membranes, collectionmembranes, and/or base together or in place, urge the separationmembranes, collection membranes, and/or base together, and allows foreasy removal of the separation and/or collection membranes from thedevice. The devices can also include identification markers (e.g.barcodes) on the base.

The devices can be used to trap and filter out solid components from aliquid biological specimen. For example, the devices can include aseparation membrane that filters and traps solid components of a wholeblood specimen (e.g. red blood cells, white blood cells, erythrocytes),thereby resulting in the collection membrane absorbing cell-free serum,plasma, and plasma proteins.

As used herein, the term “analyte” refers to any micro- ormacro-molecules in a biological specimen that are to be detected oranalyzed. These include, for example, nucleic acids (e.g. DNA, RNA),polynucleotides, oligonucleotides, proteins, polypeptides,oligopeptides, enzymes, amino acids, receptors, carbohydrates, lipids,whole cells, cellular fragments, any intra- or extra-cellular moleculesand fragments, viruses, viral molecules and fragments, bacteria, and thelike. In certain embodiments, the analytes are exogenous natural orsynthetic compounds such as small molecules like drugs, prodrugs, andmetabolites thereof. In certain embodiments, the analytes are nucleicacids such as proviral and/or viral DNA and/or RNA such as, for example,proviral and/or viral nucleic acids from human immunodeficiency virus(HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza,parvovirus B19, or any other human or animal viral pathogen. In certainembodiments, the analytes are viral particles for determining viralload. In certain embodiments, the analytes are biological markers fordetermining HLA blood types, useful for molecular diagnostic genotyping.In certain embodiments, the analytes are inflammatory biomarkers such asCXCL9/MIG and CXCL10/IP-10. In certain embodiments, the analytes aremicronutrients such as Folic Acid, Homocysteine, Retinol Binding Protein(and/or Vitamin A), Thyroglobulin, Vitamin D, trace metals (e.g. zinc),Ferritin, Transferrin Receptors, Methylmalonic Acid,Holo-Transcobalamin, C-Reactive Protein, and alpha-Acid Glycoprotein.

“Biological specimen” refers to biologic samples, either in liquid orsolid form, having contained therein an analyte of interest. Abiological specimen can be, for example, whole blood, plasma, serum,lymph, synovial fluid, bone marrow, cerebrospinal cord fluid, semen,saliva, urine, feces, sputum, vaginal lavage, skin scrapings, hair rootcells, or the like of humans or animals; physiological and pathologicalbody liquids such as secretions, excretions, exudates and transudates;any cells or cell components of humans, animals, plants, bacteria,fungi, plasmids, viruses, parasites, or the like that contain analytesof interest, and any combination thereof. In certain embodiments, abiological specimen can be a human body fluid such as whole blood, whichcan contain analytes of interest such as proviral nucleic acids and/orplasma proteins such as Troponin, monoclonal kappa and lambda free lightchains, Cystatin C, and Carbohydrate-Deficient Transferrin (CDT).

“Liquid biological specimen” means a biological fluid or a biologicalspecimen suspended in a fluid medium (e.g. water, saline, etc.).Exemplary liquid biological specimens include human, animal, plant,bacteria, fungi, plasmids, viruses, parasites (e.g. helminthes,protozoas, spirochetes) extracts or suspensions; liquid extracts orhomogenates of human or animal body tissues (e.g., bone, liver, kidney,brain); media from DNA or RNA synthesis; mixtures of chemically orbiochemically synthesized DNA or RNA; and body fluids/liquids such aswhole blood, plasma, serum, synovial fluid, cerebrospinal cord fluid,semen, and saliva.

I. Devices

Embodiments of liquid biological specimen separation devices providedherein generally comprise a base support, one or more collectionmembranes disposed on the base, one or more separation membranesdisposed on the collection membranes, and one or more covers disposed onthe one or more separation membranes, as illustrated by the non-limitingembodiments shown in FIGS. 1A-11F. One embodiment of a liquid biologicalspecimen separation device is illustrated in FIGS. 1A-2 . Anotherembodiment of a liquid biological specimen separation device isillustrated in FIGS. 3A-3B. Another embodiment of a liquid biologicalspecimen separation device is illustrated in FIGS. 4A-5 . Anotherembodiment of a liquid biological specimen separation device isillustrated in FIGS. 6-8 . Another embodiment of a liquid biologicalspecimen separation device is illustrated in FIGS. 9A-9D. Anotherembodiment of a liquid biological specimen separation device isillustrated in FIGS. 10A-10B. Another embodiment of a liquid biologicalspecimen separation device is illustrated in FIGS. 11A-11F.

FIGS. 1A-11F show a liquid biological specimen separation device 10. Theseparation device 10 can include base 12, collection membrane(s) 14,separation membrane(s) 16, cover(s) 18, collection membrane support(s)20, and/or cap(s) 32.

Base 12 generally functions as a supporting surface. Base 12 can alsofunction to secure the base 12, collection membranes 14, separationmembranes 16, covers 18, and/or collection membrane supports 20together. Base 12 can also serve as a protective enclosure that protectsany components that may be contained therein (e.g. collection membranes14, separation membranes 16, covers 18, collection membrane supports 20,biological specimens, and/or analytes enclosed by base 12) from outsideinfluences or effects.

Base 12 can assume any dimensions, size, and shape suitable for servingas a support in a liquid biological specimen separation device 10. Forexample, the general shape of base 12 can be round, rectangular, oval,square, trapezoidal, triangular, pentagonal, hexagonal, octagonal,ellipsoid, crescent, curvilinear, egg, quatrefoil, cinquefoil, and thelike. Base 12 can have a uniform shape. Base 12 can have a shape thatincludes one or more lobes/projections extending therefrom. Base 12 canhave surfaces that are uniformly flat. Base 12 can be entirely flat.Base 12 can have a three dimensional, freeform structure. Base 12 canhave one or more recesses dimensioned to receive, or projectionsdimensioned to extend into, and complementary fit collection membrane(s)14, separation membrane(s) 16, cover(s) 18, collection membranesupport(s) 20, and/or caps 32. Base 12 can have a projection extendingtherefrom shaped and dimensioned to receive or contain collectionmembrane(s) 14, separation membrane(s) 16, cover(s) 18, collectionmembrane support(s) 20, and/or caps 32.

Base 12 can have complementary features that permit separation device 10to have an open configuration and a closed configuration. For example,base 12 can have a well portion and a cap portion that assume a firstposition wherein the well portion and the cap portion extend away fromone another (open configuration) and a second position wherein the wellportion and the cap portion complementary mate with one another (closedconfiguration). As another example, base 12 can have a raised innerprojection and a cap that assume a first position wherein the raisedinner projection and the cap are separated from one another (openconfiguration) and a second position wherein the raised inner projectionand the cap complementary mate with one another (closed configuration).

Base 12 can have one or more sample deposition apertures 28 therethroughthat define an area for depositing a liquid biological specimen. Base 12can have features that improve handling and/or use of separation device10. For example, base 12 can include features, projections, tabs,handles, and the like that facilitate gripping, holding, andmanipulating separation device 10 (e.g. a tab for use in openingseparation device 10 when in a closed configuration).

Separation device 10 can have any suitable number of biological specimenreceiving/separation/collection regions such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such regions.

Base 12 can be made of any suitable material, preferably one thatprovides sufficient flexibility/stiffness and strength. Base 12 can bemade of, for example, suitable plastics materials (e.g. polyethylene,acrylic, polypropylene), paper materials (e.g. cardstock, cardboard,etc.), and the like.

Base 12 can include an identifier 22 thereon or therein. The identifier22 can be integrated/disposed into or onto an outer surface of base 12.For example, identifier 22 can be printed on or attached (e.g. adhesivelabel) to base 12. The identifier 22 can also be integrated/disposed inbase 12. The identifier 22 can use any suitable identificationtechnology. For example, identifier 22 can be any suitable RFID chip,single-dimensional (1D) barcodes, two-dimensional (2D) barcodes, QRcodes, alpha-numeric codes, and the like.

Identifier 22 can store or have associated therewith identificationinformation. Identification information can include information specificto a patient associated with a biological specimen stored therein,including personal information (address, name, sex, date of birth,ethnic background, etc.) and/or biometric information (e.g., afingerprint, a facial image or template). Identifier 22 can also storeor have associated therewith contextual information such as time, date,location of testing, and the like. To protect identifier 22 and anyinformation associated therewith, a layer of overlaminate or otherprotective material may additionally be provided over identifier 22.

One or more collection membranes 14 can be disposed on base 12. One ormore collection membrane supports 20 can also be disposed on base 12. Acollection membrane 14 can be disposed in a collection membrane support20. The collection membranes 14 generally function to absorb thefraction of a liquid biological specimen that flows through separationmembrane 16. The collection membrane supports 20 generally function tosupport (e.g. hold in place) the collection membranes 14.

Collection membranes 14 can be reversibly adhered to or held in place onbase 12 by any suitable means. For example, collection membrane 14 canbe secured in place by cover 18 (e.g. a sticker), the sticker beingadhered to base 12 and having sandwiched there between collectionmembrane 14. As another example, collection membrane 14 can be securedin place by an interference fit with another feature of separationdevice 10 (e.g. the collection membrane 14 is disposed in, and has aninterference fit with, a well in base 12). As another example,collection membrane 14 can be secured in place by disposing collectionmembrane 14 in a recess of base 12 dimensioned to fit and receivecollection membrane 14. As another example, collection membrane 14 canbe secured in place by securing collection membrane 14 in between a wellportion and a cap portion of base 12 that complementary mate with oneanother (a closed configuration). The collection membrane 14 can besecured in between the well portion and the cap portion of base 12 alongwith other features (e.g. separation membrane 16), and the stackedfeatures can be collectively dimensioned to prevent or reduce movementof the features within or out of separation device 10. As anotherexample, collection membrane 14 can be secured in place by securingcollection membrane 14 in between a raised inner projection of base 12and a cap 32 that complementary mate with one another (a closedconfiguration). The collection membrane 14 can be secured in between araised inner projection of base 12 and a cap 32 along with otherfeatures (e.g. separation membrane 16), and the stacked features can becollectively dimensioned to prevent or reduce movement of the featureswithin or out of separation device 10.

As another example, in embodiments that include collection membranesupports 20, collection membranes 12 can be connected or attached tocollection membrane supports 20, the collection membrane supports 20being adhered to, fixedly attached to, or held in place on, base 12 byany suitable means (e.g. adhesives, interference fit into a well of thebase). A benefit of this approach is that collection membranes 14 can beeasily separated from both base 12 and collection membrane supports 20.For example, the interface between a collection membrane 14 and acollection membrane support 20 can include a series of perforations thatenable easy removal of collection membrane 14 from collection membranesupport 20. This facilitates further processing of the biologicalspecimen and/or analytes of interest absorbed by collection membrane 14.

In embodiments having both collection membrane(s) 14 and collectionmembrane support(s) 20, the collection membranes 14 and collectionmembrane supports 20 can be a unitary item (e.g. a single piece ofwhatman 903 paper having a sample collection region and a supportingregion) or discrete items from one another (e.g. a first piece ofwhatman 903 paper is a collection membrane and a second piece of whatman903 paper is a collection membrane support).

The collection membrane(s) 14 and collection membrane support(s) 20 canbe made of the same material or different materials. The collectionmembranes 14 and collection membrane supports 20 can be made of anysuitable material. Suitable materials include, for example, plastics,polymers, cotton, cellulose, and/or paper. In some embodiments, thecollection membrane(s) 14 and/or collection membrane support(s) 20 arefilter papers. Filter papers that may be selected for use includecellulose fiber papers manufactured from cotton linters. Cotton linters(i.e., cotton wool) are short fibers that adhere to seeds of a cottonplant after the longer fibers have been pulled from the cotton seed.Filter papers can also include filter papers for blood collectionregistered by the U.S. Food and Drug Administration as Class II MedicalDevices (21 CFR § 862.1675), such as WHATMAN 903, AHLSTROM 142, AHLSTROM226, AHLSTROM 222, AHLSTROM 238, AHLSTROM 270, ALHSTROM 601, andESSENTRA. In some embodiments, a majority of the cellulose fibers of acellulose fiber filter paper may have sizes in the range of about 1-100microns, 10-50 microns, or 20-25 microns in length and may containnumerous hydrophobic and/or hydrophilic pockets.

Collection membranes 14 and collection membrane supports 20 can have anysuitable shape such as, for example, a circle, oval, square, rectangle,triangle, hexagonal, or other shapes and surface textures suitable foruse in the devices described herein. Collection membrane support 20 canhave a collection membrane aperture 24 for receiving collectionmembranes 14. Collection membranes 14 and collection membrane supports20 can also be dimensioned in a manner that facilitates removingcollection membranes 14 from collection membrane supports 20. Forexample, collection membrane supports 20 can include removal apertures26 that allow a device (e.g. tongs) to selectively pincer and removecollection membranes 14 from collection membrane supports 20.

Separation device 10 can include one or more collection membranes 14and/or one or more collection membrane supports 20. For example,separation device 10 can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more collection membranes 14 and/orcollection membrane supports 20. The number of collection membranes 14and the number of collection membrane supports 20 in separation device10 can be the same or they can be different. For example, in someembodiments separation device 10 can have four collection membranes 14and four collection membrane supports 20 while in other embodimentsseparation device 10 can have four collection membranes 14 and onecollection membrane support 20.

Collection membranes 14 and/or collection membrane supports 20 can haveany suitable size. In certain embodiments, collection membranes 14and/or collection membrane supports 20 can have a diameter/width of fromabout 1 mm to 50 mm, or from 10 mm to 30 mm, inclusive. In someembodiments, collection membranes 14 have a diameter/width of from about1 mm to about 15 mm.

Collection membranes 14 can include a composition absorbed to a surfacethereof, wherein the composition protects against degradation of ananalyte of interest disposed therein. Protection against degradation mayinclude protection against substantial damaging of analytes of interestcaused by chemical or biological agents including action of bacteria,free radicals, nucleases, ultraviolet radiation, oxidizing agent,alkylating agents, or acidic agents (e.g., pollutants in theatmosphere). In certain embodiments, the composition absorbed on thecollection membrane 14 can include one or more of a weak base, achelating agent, a protein denaturing agent such as a detergent orsurfactant, a nuclease inhibitor, a free radical trap, and an oxygenscavenger element. As used herein, a “weak base” can be a Lewis basewhich has a pH of about 6 to 10, preferably about pH 8 to 9.5. In a casewhere the stored analyte of interest is RNA, particularly unstable RNA,the composition may include RNase inhibitors and inactivators, geneticprobes, complementary DNA or RNA (or functionally equivalent compounds),proteins and organic moieties that stabilize RNA or prevent itsdegradation.

One or more separation membranes 16 can be disposed on base 12,collection membranes 14, covers 18 and/or collection membrane supports20. The separation membranes 16 generally function to separate a liquidbiological specimen into a first fraction and a second fraction. Thefirst fraction is contained/trapped within separation membranes 16 whilethe second faction passes through separation membranes 16 and intocollection membranes 14.

Separation membranes 16 can be reversibly held in place on base 12,collection membranes 14, covers 18, and/or collection membrane supports20 by any suitable means. For example, separation membranes 16 can besecured in place by cover 18 (e.g. a sticker), the sticker being adheredto base 12 and/or collection membrane supports 20 and having sandwichedthere between collection membranes 14, collection membrane supports 20,and/or separation membranes 16. The compressive force keeps separationmembranes 16 in contact with collection membranes 14. In anotherexample, separation membranes 16 can be secured in place by cover 18(e.g. a sticker), the sticker being adhered to base 12 and havingsandwiched there between separation membranes 16. In another example,separation membranes 16 can be secured in place by an interference fitwith another feature of separation device 10 (e.g. the separationmembrane 16 is disposed in, and has an interference fit with, a well inbase 12). As another example, separation membrane 16 can be secured inplace by disposing separation membrane 16 in a recess of base 12dimensioned to fit and receive separation membrane 16.

As another example, separation membrane 16 can be secured in place bysecuring separation membrane 16 in between a well portion and a capportion of base 12 that complementary mate with one another (a closedconfiguration). The separation membrane 16 can be secured in between thewell portion and the cap portion of base 12 along with other features(e.g. collection membrane 14), and the stacked features can becollectively dimensioned to prevent or reduce movement of the featureswithin or out of separation device 10. As another example, separationmembrane 16 can be secured in place by securing separation membrane 16in between a raised inner projection of base 12 and a cap 32 thatcomplementary mate with one another (a closed configuration). Theseparation membrane 16 can be secured in between a raised innerprojection of base 12 and a cap 32 along with other features (e.g.collection membrane 14), and the stacked features can be collectivelydimensioned to prevent or reduce movement of the features within or outof separation device 10.

Both collection membranes 14 and separation membranes 16 are easilyseparable from separation device 10 so that collection membranes 14 andseparation membranes 16 can be accessed and removed from separationdevice 10 for further analysis.

Separation membranes 16 are generally made of a material that allows forflow of a liquid biological specimen or a fraction thereof therethrough.Separation membranes 16 can comprise a plurality of fibers. Separationmembranes 16 can be made of a material that has a gradually decreasingpore size (e.g. an asymmetric porous membrane) from a top side (e.g. theside where biological specimens are initially deposited) to a bottomside (the side in contact with collection membrane 14). Separationmembrane 16 can also be made of a material that has a uniform pore sizethroughout. In certain embodiments, flow of a liquid biological specimendeposited on separation membrane 16 through separation membrane 16 isdriven by capillary forces (e.g. capillary flow) and/or gravity. Incertain embodiments, materials suitable for use in separation membranes16 are those in which one biological specimen moves faster through theseparation membrane than another biological specimen (e.g. blood plasmamoves faster than corpuscles).

Suitable materials for use in separation membranes 16 can include, forexample, synthetic polymers having fine fiber diameter and fibers madeof glass or porous polymers. In certain preferred embodiments,separation membranes 16 are made of a polysulfone polymer materialhaving a porosity that gradually decreases from a top side of themembrane to a bottom side of the membrane so as to filter and trap solidand/or liquid components of a liquid biological specimen deposited onseparation membranes 16. Separation membrane materials can include, forexample, synthetic or natural polymers such as cellulose mixed esters,polyvinylidene difluoride, polytetrafluoroethylene, polycarbonate,polypropylene, polyester, and polysulfone polymers and matrices (e.g.,asymmetric sub-micron polysulfone (BTS) and/or asymmetric super micronpolysulfone (MMM) made by Pall Corporation). Separation membranematerials can also include, for example, VIVID GR, VIVID GX, and CYTOSEP1660. A person of ordinary skill will readily appreciate that othermembranes or filtering materials can be used. In some embodiments,separation membranes 16 are suitable for blood component filtering andserum/plasma separation. In some embodiments, separation membranes 16have a porosity of not more than 30%, and preferably not more than 25%.In certain embodiments, separation membranes 16 can be made ofpolysulfone polymer having a pore size ranging from about 0.1-20 micronsand a pore size ratio from about 50:1 to 100:1.

The size of a separation membrane 16 can be larger than the size of acollection membrane 14 to which it is removably disposed upon. Forexample, the size of a separation membrane 16 may be at least 20%, or atleast 30%, or at least 40%, or at least 50% larger than a size of acorresponding collection membrane 14. Alternatively, the size of aseparation membrane 16 can be the same or about the same (e.g. within10% by area) size as a collection membrane 14 to which it is removablydisposed upon. Alternatively, the size of a separation membrane 16 canbe smaller than the size of a collection membrane 14 to which it isremovably disposed upon. In certain embodiments, a separation membrane16 has a diameter/width of from about 1 mm to 50 mm, or from 10 mm to 30mm, inclusive. For example, separation membrane 16 can have adiameter/width of about 10 mm to about 20 mm.

Separation membrane 16 can have a shape that is the same shape as acorresponding collection membrane 14 (e.g. circles). Separation membrane16 can also have a shape that is different from a shape of acorresponding collection membrane 14 and, thus, does not align in itsentirety with the shape of collection membrane 14 when brought intocontact thereto. For example, separation membrane 16 can have anirregular or oblong shape (e.g., a racquet shape with a handle-likeextension extending on a lateral side thereof) whereas collectionmembrane 14 can have a circular shape.

Separation device 10 can include one or more separation membranes 16.For example, separation device 10 can include 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more separation membranes16.

One or more covers 18 can be disposed on separation membranes 16,collection membranes 14, collection membrane supports 20, and/or base12. The covers 18 generally have one or more sample deposition apertures28 therethrough that define an area for depositing a liquid biologicalspecimen. The covers 18 can also function to secure the separationmembranes 16, collection membranes 14, collection membrane supports 20,and/or base 12 together. Covers 18 are reversibly adhered or attached toseparation membranes 16, collection membranes 14, collection membranesupports 20, and/or base 12 by any suitable means. For example, covers18 can be stickers having an adhesive side that is used to attach covers18 to separation membranes 16, collection membranes 14, collectionmembrane supports 20, and/or base 12. Covers 18 are reversibly adheredso that covers 18 are easily removed in order to access and removecollection membranes 14 and/or separation membranes 16 from separationdevice 10 for further processing and analysis.

Covers 18 can include one or more projections 30 that make removal ofcovers 18 easier. For example, covers 18 can have a graspable tab tofacilitate removal of covers 18 from separation device 10.

Separation device 10 can also include a desiccant for drying, or keepingdry, a biological specimen. The desiccant can be disposed anywhere onseparation device 10 (e.g. on or in base 12).

Separation device 10 can include one or more caps 32. Caps 32 generallyhave one or more sample deposition apertures 28 therethrough that definean area for depositing a liquid biological specimen. Caps 32 can operateto removably secure the separation membranes 16, collection membranes14, and/or base 12 together. Caps 32 can be reversibly attached to base12 by any suitable means. For example, separation device 10 can includeelastomeric band fasteners 38 that urge and hold base 12 and caps 32together. As another example, base 12 can be secured to cap 32 by atwist-lock mechanism 54 that urges and holds base 12 and caps 32together. Caps 32 can be made of, for example, suitable plasticsmaterials (e.g. polyethylene, acrylic, polypropylene).

FIGS. 1A-2 show a liquid biological specimen separation device 10 inaccordance with one embodiment of the invention. The liquid biologicalspecimen separation device 10 has a base 12. Base 12 is a flat bifacialsheet having a uniform thickness and a modified cinquefoil shape definedby a series of radial projections extending therefrom. The largestradial projection is a handle that facilitates handling/holdingseparation device 10. Each of the four remaining radial projectionsprovide a supporting surface or region for a corresponding one of thebiological specimen receiving/separation/collection regions.

Disposed on liquid biological specimen separation device 10 are fourcircular collection membrane supports 20 and corresponding circularcollection membranes 14 disposed therein. Each of the four collectionmembrane supports 20 is separated from a corresponding collectionmembrane 14 by perforations, the perforations enabling easy removal ofthe collection membranes 14 from their respective collection membranesupport 20. Each of the collection membrane supports 20 also includestwo removal apertures 26 that enable the use of a device (e.g. tweezersor tongs) to selectively grasp and remove collection membranes 14 fromtheir respective collection membrane support 20.

The liquid biological specimen separation device 10 has four circularseparation membranes 16, each of which is disposed on a respective oneof the collection membranes 14 and collection membrane supports 20. Thecircular separation membranes 16, when disposed on collection membranes14, completely overlap the collection membranes 14 because the diameterof separation membranes 16 is larger than the diameter of collectionmembranes 14. The diameter of separation membranes 16, however, issmaller than the diameter of collection membrane supports 20.

The liquid biological specimen separation device 10 has four generallycircular covers 18, each having a tab extending therefrom. The covers 18are stickers, and each sticker is adhesively disposed on a respectiveone of the separation membranes 16 and collection membrane supports 20.Each sticker has a biological sample deposition aperture extendingtherethrough that permits depositing a liquid biological specimen ontothe separation membrane 16 secured underneath the sticker.

FIGS. 3A-3B show a liquid biological specimen separation device 10 inaccordance with another embodiment of the invention. The liquidbiological specimen separation device 10 of FIGS. 3A-3B is substantiallythe same as the liquid biological specimen separation device 10 of FIGS.1A-2 except that base 12 is a semi-circular shape having one radialprojection extending therefrom, the radial projection serving as ahandle for handling separation device 10.

FIGS. 4A-5 show a liquid biological specimen separation device 10 inaccordance with another embodiment of the invention. The liquidbiological specimen separation device 10 of FIGS. 4A-5 is similar to theliquid biological specimen separation device 10 of FIGS. 1A-2 with a fewdifferences. As shown in FIGS. 4A-5 , separation device 10 has onecollection membrane support 20 and one cover 18. The collection membranesupport 20 and cover 18 are generally shaped to match the portion ofbase 10 having the four radial projections. Cover 18 also has two tabsextending therefrom and projecting outwards from separation device 10.

FIGS. 6-8 show a liquid biological specimen separation device 10 inaccordance with another embodiment of the invention. In this embodiment,base 12 is a generally square shape having four radial lobes projectingoutwards from separation device 10, one projection at each corner. Theliquid biological specimen separation device 10 includes one collectionmembrane 14, one collection membrane support 20, one separation membrane16, and one cover 18.

FIGS. 9A-9D show a liquid biological specimen separation device 10 inaccordance with another embodiment of the invention. The liquidbiological specimen separation device 10 has a base 12. Base 12 is amade of a clear plastic material and has a flip top design. Base 12 hasfour wells 30 and four corresponding caps 32 that are designed tocomplementary mate with one another. Caps 32 are connected to the restof base 12 by arms 34. Arms 34 have a hinge 36 that allows movement ofcaps 32 relative to wells 30. Base 12 can assume a first positionwherein caps 32 extend away from wells 30 (e.g. an open configuration,as shown). Base 12 can assume a second position wherein caps 32 andwells 30 complementary mate with one another to form a closedconfiguration. Each cap 32 includes a sample deposition aperture 28therethrough that defines an area for depositing a liquid biologicalspecimen. Each cap 32 also includes a tab feature that facilitatesopening separation device 10 when in a closed configuration.

Disposed in wells 30 of liquid biological specimen separation device 10are four circular collection membrane supports 20. Circular collectionmembranes 14 (not shown) are disposable in a respective one of thecollection membrane supports 20. Each of the collection membranesupports 20 includes two removal apertures 26 that enable the use of adevice (e.g. tweezers or tongs) to selectively grasp and removecollection membranes 14 from their respective collection membranesupport 20.

Four circular separation membranes 16 (not shown) are disposable in arespective one of the recesses in caps 32. Four generally circularcovers 18 (not shown) are disposable on a respective one of theseparation membranes 16. The covers 18 are stickers, and each sticker isadhesively disposed on a respective one of the separation membranes 16and an internal surface of caps 32. Each sticker adheres a separationmembrane 16 to an internal surface of a respective cap 32. Each stickerhas a biological sample deposition aperture extending therethrough thatpermits flow from separation membrane 16 to collection membrane 14. In aclosed configuration of separation device 10, at least a portion ofseparation membrane 16 comes into contact with collection membrane 14(some contact between the two membranes can be intermediated by a cover18).

FIGS. 10A-10B show a liquid biological specimen separation device 10 inaccordance with another embodiment of the invention. The liquidbiological specimen separation device 10 has a base 12. Base 12 is amade of a plastic material. Base 12 has a three dimensional shapecomprising a relatively low profile outer periphery 40 and a raisedinner projection 42. The raised inner projection 42 has a first recess44 therein dimensioned to receive a separation membrane 16. The firstrecess 44 has a second recess 46 therein dimensioned to receive acollection membrane 14. Base 12 also has two fastening tabs 50 extendingtherefrom.

The liquid biological specimen separation device 10 has a cap 32. Thecap 32 is dimensioned to complementary mate with raised inner projection42. Cap 32 includes a funnel portion arranged to direct fluid flowtowards a sample deposition aperture 28 extending through cap 32, thesample deposition aperture 28 defining an area for depositing a liquidbiological specimen. Cap 32 has two diametrically opposing pegs 48extending therefrom. Pegs 48 are positioned on cap 32 such that they arealignable with fastening tabs 50.

Disposed in the second recess 46 is a collection membrane 14. The secondrecess 46 and collection membrane 14 can be dimensioned such thatcollection membrane 14 sits flush with a top surface of the first recess44. The second recess 46 and collection membrane 14 can be dimensionedsuch that that collection membrane 14 extends a short distance (e.g. 0.5mm) above a top surface of the first recess 44. In either case, at leasta portion of separation membrane 16 comes into contact with collectionmembrane 14.

Disposed in the first recess 44 is a separation membrane 16. Separationmembrane 16, collection membrane 14, and cap 32 are aligned about anaxis extending through a center point of each of the separation membrane16, collection membrane 14, and cap 32.

The liquid biological specimen separation device 10 has two fasteners38. Fasteners 38 are elastomeric bands wrappable around pegs 48 andfastening tabs 50 such that base 12 is secured to cap 32.

The liquid biological specimen separation device 10 has at least aclosed configuration and an open configuration.

In a closed configuration, fasteners 38 are wrapped around pegs 48 andfastening tabs 50, thereby securing base 12 to cap 32. Contained inbetween base 12 and cap 32 is collection membrane 14, which is disposedin the second recess 46, and separation membrane 16, which is disposedin the first recess 44. Fasteners 38 urge cap 32 towards base 12 withsufficient force such that cap 32 collapses pores in separation membrane16, thereby forming a liquid impermeable barrier 52 in between tworegions of separation membrane 16. The liquid impermeable barrier 52 isdimensioned to be slightly larger in width (e.g. radius) than collectionmembrane 14, thereby minimizing the volume of plasma lost to lateralleakage and/or absorption by separation membrane 16.

In an open configuration, fasteners 38 are not wrapped around pegs 48and fastening tabs 50, thereby allowing disassembly of the liquidbiological specimen separation device 10. For example, collectionmembrane 14 and separation membrane 16 are removable from liquidbiological specimen separation device 10 for use in various analytictechniques.

Sample deposition aperture 28 can have a diameter/width of 8.3 to 9.2mm. That is, sample deposition aperture 28 can have a diameter/width of8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, or 9.2 mm.

Collection membrane 14 can have a diameter/width of 3 to 10.5 mm and athickness of 0.19 mm to 0.270 mm.

First recess 44 can have a diameter/width of 10 to 25 mm.

Separation membrane 16 can have a diameter/width of 9 to 11 mm and athickness of 0.19 mm to 0.380 mm.

Second recess 46 can have a diameter/width that is 0.1 to 0.5 mm largerthan a diameter/width of collection membrane 14, and preferably has adiameter/width that is smaller than the sample deposition aperture 28.

Liquid impermeable barrier 52 can have a diameter/width that is 0.6 to1.0 mm larger than a diameter of sample deposition aperture 28.

FIGS. 11A-11F show a liquid biological specimen separation device 10 inaccordance with another embodiment of the invention. The liquidbiological specimen separation device 10 of FIGS. 11A-11F issubstantially the same as the liquid biological specimen separationdevice 10 of FIGS. 10A-10B except that base 12 is secured to cap 32 by atwist-lock mechanism 54 rather than fasteners 38, pegs 48, and fasteningtabs 50. Base 12 and cap 32 each have complementary fasteningprotrusions 56 with ridge features 58. The complementary fasteningprotrusions 56 of base 12 and cap 32 can be engaged to secure base 12 tocap 32 and disengaged to unsecure base 12 from cap 32, respectively, byrotating base 12 relative to cap 32. When the complementary fasteningprotrusions 56 of base 12 and cap 32 are engaged with one another, cap32 is urged towards base 12 with sufficient force such that cap 32collapses pores in separation membrane 16, thereby forming a liquidimpermeable barrier 52 in between two regions of separation membrane 16.

In operation, to secure cap 32 to base 12, complementary fasteningprotrusions 56 on cap 32 can be inserted into apertures 60 in base 12that are dimensioned to receive complementary fastening protrusions 56.Cap 32 is then rotated relative to base 12 such that complementaryfastening protrusions 56 and ridge features 58 on both base 12 and cap32 are aligned and engaged. The reverse operations can be performed tounsecure cap 32 from base 12.

I. Applications and Use

Methods of using a liquid biological specimen separation device areprovided. Generally, a liquid biological specimen separation device isused to receive a liquid biological specimen containing an analyte ofinterest, separate the liquid biological specimen into two components,and store an analyte of interest. The liquid biological specimenseparation device is suitable for use as a point-of-care device.

In certain embodiments, the methods include providing a liquidbiological specimen separation device and dispensing a liquid biologicalspecimen onto a separation membrane of the device via an aperture in acover and/or a cap. The liquid biological specimen flows through theseparation membrane (e.g. via capillary action, gravity, etc). A firstcomponent of the liquid biological specimen is trapped/retained by theseparation membrane while a second component of the liquid biologicalspecimen flows through the separation membrane and into the collectionmembrane, which absorbs the second component. The second component canbe dried in the collection membrane, either actively (e.g. via adesiccant or heating) or passively (e.g. air dry), before furtherprocessing. Alternatively, the second component can be used for furtherprocessing prior to being dried out (e.g. while still wet). Theseparation membrane and/or the collection membrane having the first andsecond components respectively can be removed from the device and beexposed to or placed in a reconstitution media to remove/recoveranalytes of interest therefrom, which can then be analyzed using asuitable technique for the analyte to be studied. In certainembodiments, the methods can include compressing the collection and/orseparation membranes to aid in recovering analytes of interesttherefrom. In certain embodiments, the methods can include applyingreconstitution media to the separation and/or collection membranes torehydrate analytes of interest contained therein, and compressing themembranes to release the analytes of interest. In certain embodiments,the separation devices and methods separate a liquid biological specimeninto at least two constituent components that are subsequently air-driedand stored at ambient temperatures (e.g. without the need forrefrigeration or freezing) prior to subsequent quantitative andqualitative analysis on at least one analyte of interest in at least oneof the components.

In certain embodiments, the methods include providing a liquidbiological specimen separation device. The methods can include addingapproximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200 uL of whole blood from either a pipetteor directly from a patient's finger-stick or heel-stick onto a topsurface of a separation membrane of the device via an aperture in acover and/or a cap. In embodiments, disposing 20-200 uL of whole bloodonto a top surface of a separation membrane of the device results in5-50 uL of plasma saturating a collection membrane. In embodiments, thevolume of plasma is equal to about one-quarter the volume of wholeblood. The methods can include adding approximately 25-40 uL of wholeblood from either a pipette or directly from a patient's finger-stick orheel-stick onto a top surface of a separation membrane of the device viaan aperture in a cover and/or a cap. In embodiments, disposing 25-40 uLof whole blood onto a top surface of a separation membrane of the deviceresults in 6-10 uL of plasma saturating a collection membrane.

The methods can include having a user wait approximately 1, 2, 3, 4, 5,or 10 or more minutes for transfer of plasma through the separationmembrane to the collection membrane (e.g. via capillary action, gravity,etc). The methods can include having a user allow the collectionmembrane to dry (e.g. placing the collection membrane in a designatedair drying location) for 1, 2, 3, 4, 5, 6, 12, 24, or more hours. Thedrying can be accomplished at room temperature by air drying or atcontrolled temperature. The methods can include placing the whole deviceor the dried collection membrane in a sealable packaging, which caninclude a desiccant, for shipment to a laboratory for further analysis.The methods can include the laboratory separating the collectionmembrane from the separation device. The methods can include thelaboratory separating white blood solids from the collection membrane.The methods can include the laboratory suspending the collectionmembrane in a reconstitution medium.

In certain embodiments, the reconstitution medium is molecular-gradewater. In other embodiments, the reconstitution medium includesnuclease-free water or the components of phosphate buffered saline (PBS)or other suitable buffered saline solutions. Optionally, thereconstitution medium includes sodium azide or other antimicrobialagents. The reconstitution medium can also include any number orcombinations of available biological preservatives or bloodanticoagulants including but not limited to ethylenediaminetetraaceticacid (EDTA), sodium citrate, and heparin. Saline solutions ornuclease-free water can serve as a sterile and neutral medium for therehydration, re-suspension, and recovery of analyte(s) of interest fromthe collection and/or separation membranes. When included, antimicrobialagents such as sodium azide prevent microbial growth and subsequentcontamination with RNases. When included, biological preservatives suchas EDTA, sodium citrate, and heparin serve as anticoagulants and orchelating agents.

The volume of a membrane may or may not expand upon absorption of aliquid biological specimen, and may or may not contract upon drying.However, a liquid saturated membrane can be compressed to releaseentrained fluid containing an analyte of interest, due to its porosity,by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, ormore of its saturated volume. Volumetric compression is one convenienttechnique for releasing analytes of interest, however, other means suchas centrifugation or vacuum pressure can alternatively be employed torelease analytes of interest from a membrane.

In certain embodiments, the methods can also include an intermediatestep of applying a stabilizing composition to the collection membraneand/or the separation membrane to protect analytes of interest againstdegradation. Depending upon the analytes of interest, the stabilizingcomposition may include one or more of a weak base, a chelating agent, aprotein denaturing agent such as a detergent or surfactant, a nucleaseinhibitor, and a free radical trap. Particularly for protection ofunstable RNA, the stabilizing composition may include RNase inhibitorsand inactivators, genetic probes, complementary DNA or RNA (orfunctionally equivalent compounds), proteins and organic moieties thatstabilize RNA or prevent its degradation.

In embodiments, the time periods for which analytes of interest may bepreserved or stored on a collection and/or separation membrane can befor a period of several minutes, hours, days, months, or even greater.

Temperature conditions under which analytes of interest may be preservedor stored on a collection and/or separation membrane are not limited.Typically, analytes of interest are kept at ambient or room temperature,for example, from about 15° C. to about 40° C., preferably from about15° C. to about 25° C. In some embodiments, the analytes of interest maybe kept in a cool environment. For example, in short-term storage, theanalytes can be refrigerated at about 2° C. to about 10° C. In yetanother example, the analytes may be refrigerated at about 4° C. toabout 8° C. In another example, in long-term storage, the analytes canbe frozen at about −20° C. to about −80° C. In addition, the membranesmay preferably, but not necessarily, be stored in dry or desiccatedconditions or under an inert atmosphere.

In certain embodiments, whole blood is dispensed onto a liquidbiological specimen separation device. In such embodiments, whole bloodor a liquid suspension thereof is deposited onto a separation membrane.The separation membrane absorbs the whole blood. The separation membranecaptures some solid components of whole blood (e.g., WBCs, RBCs,platelets, and/or other cellular components) while allowing fluidicand/or other solid whole blood components (e.g. cell-free plasma) topass through the separation membrane via gravity and/or capillaryaction. The components of whole blood passing through the separationmembrane are absorbed by the collection membrane.

In certain embodiments, cell-free plasma captured on the collectionmembrane can be removed/recovered from the collection membrane byexposing the collection membrane to a reconstitution media. Therecovered cell-free plasma can contain an analyte of interest, forinstance, nucleic acids such as DNA and RNA, which can be used for viralload quantitation, genotyping, drug resistance testing, or othersuitable analyses. The analytes of interest can be detected or analyzedusing analytical and diagnostic methods known in the art.

The detailed description set forth above is provided to aid thoseskilled in the art in practicing the invention. However, the inventiondescribed and claimed herein is not to be limited in scope by thespecific embodiments described above, as these embodiments are presentedas mere illustrations of several aspects of the invention. Anycombinations and modifications of the described methods and components,and compositions used in the practice of the methods, in addition tothose not specifically described, will become apparent to those skilledin the art based on the present disclosure and do not depart from thespirit or scope of the present invention. Such variations,modifications, and combinations are also encompassed by the presentdisclosure and fall within the scope of the appended claims.

What is claimed is:
 1. A liquid biological specimen separation devicecomprising: a base; a well raised away from the base; a collectionmembrane disposed on the well; a separation membrane disposed on thecollection membrane; a cap disposed on the separation membrane andremovably complementary mated with the well in a closed configuration,wherein the cap comprises an aperture therein configured to allowdeposition of a liquid biological specimen therethrough; and at leasttwo elastomeric band fasteners, wherein the cap removably adheres to thebase by the at least two elastomeric band fasteners, wherein the cap isremovable from the well to change from the closed configuration into anopen configuration, and wherein the collection membrane and theseparation membrane are secured between the cap and the well in theclosed configuration.
 2. The device of claim 1, wherein the separationmembrane has a porosity that gradually decreases from a first side to asecond side so as to filter and trap solid components of a liquidbiological specimen deposited on the separation membrane.
 3. The deviceof claim 1, wherein the separation membrane is configured to filter andtrap solid components of a biological specimen, the biological specimenbeing selected from the group consisting of whole blood, plasma, urine,saliva, sputum, semen, vaginal lavages, bone marrow and cerebrospinalfluid.
 4. The device of claim 1, wherein the separation membrane isconfigured to filter and trap solid components of a whole bloodspecimen, and wherein the collection membrane is configured toseparately filter and trap a plasma fraction or filtrate of the wholeblood specimen.
 5. The device of claim 1, wherein the separationmembrane has a pore size ranging from 0.1-20 μm.
 6. The device of claim1, wherein the separation membrane comprises a polysulfone polymermaterial selected from the group consisting of asymmetric sub-micronpolysulfone and asymmetric super micron polysulfone.
 7. The device ofclaim 1, wherein the collection membrane comprises a substantiallyhydrophobic polyolefin material comprising a plurality of polypropylenefibers coated with hydrophobic polyethylene.
 8. The device of claim 1,wherein the collection membrane, the separation membrane, or both,comprise microglass fibers.
 9. The device of claim 1, wherein the capremovably adheres to the base.
 10. The device of claim 1, wherein thecap is urged towards the base.
 11. The device of claim 10, wherein theseparation membrane comprises a liquid impermeable barrier between tworegions of the separation membrane formed from collapsed pores in theseparation membrane as a result of the urging.
 12. The device of claim1, further comprising an identifier disposed on the base.
 13. The deviceof claim 1, wherein the well comprises a first recess and a secondrecess.
 14. The device of claim 13, wherein the first recess and secondrecess are dimensioned to receive the separation membrane and thecollection membrane, respectively.
 15. The device of claim 13, whereinthe second recess is disposed in the first recess.
 16. The device ofclaim 1, wherein the aperture, the separation membrane, and thecollection membrane are aligned about an axis extending through a centerpoint of each of the aperture, the separation membrane, and thecollection membrane in the closed configuration.
 17. A method forseparating plasma from whole blood comprising: providing a deviceaccording to claim 1; and depositing whole blood through the apertureand onto the separation membrane.
 18. The method of claim 17, wherein25-40 uL of whole blood is deposited onto the separation membrane. 19.The method of claim 17, wherein 6-10 uL of plasma are recovered in thecollection membrane.